Recently, a large number of measurements using intermolecular interactions such as immune responses are being carried out in clinical tests, etc. However, since conventional methods require complicated operations or labeling substances, several techniques are used that are capable of detecting the change in the binding amount of a test substance with high sensitivity without using such labeling substances. Examples of such a technique may include a surface plasmon resonance (SPR) measurement technique, a quartz crystal microbalance (QCM) measurement technique, and a measurement technique of using functional surfaces ranging from gold colloid particles to ultra-fine particles. The SPR measurement technique is a method of measuring changes in the refractive index near an organic functional film attached to the metal film of a chip by measuring a peak shift in the wavelength of reflected light, or changes in amounts of reflected light in a certain wavelength, so as to detect adsorption and desorption occurring near the surface. The QCM measurement technique is a technique of detecting adsorbed or desorbed mass at the ng level, using a change in frequency of a crystal due to adsorption or desorption of a substance on gold electrodes of a quartz crystal (device). In addition, the ultra-fine particle surface (nm level) of gold is functionalized, and physiologically active substances are immobilized thereon. Thus, a reaction to recognize specificity among physiologically active substances is carried out, thereby detecting a substance associated with a living organism from sedimentation of gold fine particles or sequences.
In all of the above-described techniques, the surface where a physiologically active substance is immobilized is important. Surface plasmon resonance (SPR), which is most commonly used in this technical field, will be described below as an example.
A commonly used measurement chip comprises a transparent substrate (e.g., glass), an evaporated metal film, and a thin film having thereon a functional group capable of immobilizing a physiologically active substance. The measurement chip immobilizes the physiologically active substance on the metal surface via the functional group. A specific binding reaction between the physiological active substance and a test substance is measured, so as to analyze an interaction between biomolecules.
As an example of a detection surface having a functional group by which a physiologically active substance can be immobilized, JP Patent No. 2815120 discloses in detail a method for producing hydrogel. Specifically, a barrier layer is formed by the binding of 16-mercaptohexadecanol layer to a gold film. On the gold film, the hydroxyl group of the barrier layer is epoxy-activated by treatment with epichlorohydrin. At the next stage, dextran is adhered to the barrier layer via ether linkage. Next, bromoacetic acid is reacted with the dextran matrix, thereby introducing a carboxymethyl group.
The following technique has been disclosed as techniques for immobilizing a physiologically active substance (e.g., protein or amino acid) having an amino group on the surface of the carboxymethyl-modified dextran produced based on this method. Namely, some carboxyl groups of the carboxymethyl-modified dextran are modified by treatment with an aqueous solution of N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) hydrochloric acid, so as to generate reactive ester functions, for example. Residual charges (that is, unreacted carboxyl groups) will contribute to the achievement of condensation of the physiologically active substance on a detection surface. By allowing an aqueous solution of a physiologically active substance (protein or amino acid) containing an amino group to come into contact with such detection surface, the physiologically active substance containing an amino group can be bound to a dextran matrix via covalent bonding.
Hydrogel produced by the above-mentioned method exerts excellent performance as the detection surface of a biosensor, because a physiologically active substance containing an amino group can be three-dimensionally immobilized. However, the method for producing hydrogel according to the above-mentioned method is problematic in that the method is complicated and the required production time is long. Furthermore, the method is also problematic in terms of safety because it requires the use of compounds such as epichlorohydrin or bromoacetic acid.
On the other hand, as a typical technique for immobilizing a physiologically active substance on a measurement chip, a method (amine coupling method) that involves binding an amino group of a physiologically active substance to a carboxyl group on a measurement chip is broadly used. This method requires dissolving a physiologically active substance in a buffer having a pH that is lower than the isoelectric point of such substance upon immobilization. Specifically, whereas a physiologically active substance will be positively charged when the pH is the isoelectric point or lower, the carboxyl group on a measurement chip are negatively charged from the alkali side to the acidic region of approximately pH 3.5. Therefore, a physiologically active substance is concentrated on a measurement chip due to electrostatic attraction. When such preconcentration does not occur, the immobilization amount of a physiologically active substance will drastically decrease. Thus, a physiologically active substance to be immobilized should be dissolved in a buffer having a pH that is lower than the isoelectric point of such substance, as disclosed in J. C. S. Chem. Commun., 1990, 1526 and U.S. Pat. No. 5,436,161.
This means that a physiologically active substance that is denatured under low-pH conditions is unable to be immobilized while maintaining its activity. Furthermore, a physiologically active substance such as an acidic protein has no positive net charge, even in the case of a pH of approximately 3.5. Thus, no preconcentration effects can be obtained, so that immobilization becomes impossible.
A physiologically active substance dissolved in a buffer having a pH that is higher than the isoelectric point can be immobilized on a solid surface because of electrostatic attraction between the substance and a cationic polymer immobilized on the solid surface. JP Patent Publication (Kokai) No. 8-245815 A (1996) discloses a technique using such principle, which involves alternately layering a protein and an organic polymer ion.
This method is very excellent in that a physiologically active substance can be conveniently immobilized. However, two problems arise in view of application to a biosensor. The first problem is that because binding between a protein and a substrate depends only on electrostatic interaction, a part of the physiologically active substances that have been electrostatically adsorbed on a solid surface may be dissociated due to a washing step using an acidic solution or an alkaline solution. The second problem is that a physiologically active substance is obtained as a densely packed monomolecular layer. In order to increase the immobilization amount of a physiologically active substance, it is desirable to three-dimensionally immobilize a physiologically active substance. Furthermore, dense packing of a physiologically active substance is not preferable in terms of application to a biosensor for measuring binding and dissociation behaviors of a compound interacting with a physiologically active substance.
Further, in the case of a measurement chip having a carboxyl group, such as a measurement chip having carboxymethyl dextran immobilized thereon as described in J. C. S. Chem. Commun., 1990, 1526, it is difficult to immobilize a protein, because preconcentration does not occur at pH 3.5 that is the acid dissociation constant of a carboxyl group, or lower.
In the amine coupling method as mentioned above, a measurement chip is previously coated with a polymer for protein immobilization and then a physiologically active substance is bound to the polymer. Thus, the physiologically active substance can be immobilized on the measurement chip. As a polymer for protein immobilization, a carboxymethylated (—CH2COOH) polymer is known (U.S. Pat. No. 5,436,161, Colloids and Surfaces B: Biointerfaces, 1, 1993, 83-89; and Biosensors and Bioelectronics, 10, 1995, 813-822). However, this polymer has an anionic group in its molecule. Thus, the pH of a protein solution should be adjusted at the isoelectric point or lower, in order to immobilize a protein using charge concentration. Hence, an acidic protein should be immobilized in a low-pH region. Furthermore, such fluid is problematic in that it causes lower protein activity or death. Furthermore, when introduction of a cationic group into such polymer is attempted, a problem arises in that the cationic group and the carboxyl group that is a reactive group for protein immobilization form a salt and the polymer is gelatinized, so that the protein can not be immobilized.